Fundamental properties shared by all stem cells (e.g., self renewal, cell type-specific propagation) are likely to be regulated by overlapping molecular pathways. To date, however, relatively few genes have been identified that are associated with stem cell formation or maintenance, due partly to technical limitations in purifying mammalian stem cells to homogeneity. The glossiphoniid leech, Theromyzon rude, offers a particularly well-suited experimental system for examining the molecular aspects of stem cell genesis and differentiation. Leech embryonic stem (LES) cells and their founder cells (precursors) are among the largest embryonic cells in the animal kingdom (50 - 400 um for Theromyzon); their large size and asymmetrical distribution on the surface of developing embryos has permitted their identification and homogeneous isolation. This proposal seeks to characterize genes that are differentially-expressed at the precursor to LES cell transition. By Differential Display and Northern blot analyses, dynamic changes in gene expression have been identified in LES cells and their immediate cell precursors, respectively. In situ hybridization analysis will be conducted on a selected group of cDNAs to establish their spatiotemporal expression patterns prior to and following LES cell formation. In an effort to establish the specific roles of candidate genes in LES cell genesis, gene manipulation experiments will be conducted during the early stages of development using antisense oligonucleotides, RNA interference (RNAi), and direct micro-injections of engineered plasmid and mRNA constructs. Preliminary studies with gene-specific antisense oligonucleotides have generated phenotypes ranging from LES cell deficiencies to the abnormal distribution and proliferation of LES cell progeny. It is anticipated that LES cell-associated genes characterized in this study will have mammalian counterparts, based upon the conservation of developmentally important genes across metazoan phyla.